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Khalil RA. Regulation of Vascular Smooth Muscle Function. San Rafael (CA): Morgan & Claypool Life Sciences; 2010.

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Regulation of Vascular Smooth Muscle Function.

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Chapter 6Protein Kinase C

Because of the small size and diffusible nature of Ca2+, it has been feasible to envision its role in transducing the extracellular signal to the VSM contractile myofilaments. In contrast, the role of PKC in VSM contraction is not as widely perceived as Ca2+ partly because of its relatively large size, its numerous isoforms and substrates, and its differential subcellular distribution during VSM activation. Some important questions are how PKC is identified among other kinases in VSM and how the PKC signal is transferred from the receptors at the cell surface to the contractile myofilaments in the center of the cell. This section will discuss PKC structure, isoforms, protein substrates, subcellular distribution, and its potential role as a modulator of VSM function.


PKC is an ubiquitous enzyme that was originally described as a Ca2+-activated, phospholipid- dependent protein kinase [123]. Molecular cloning and biochemical analysis have revealed a family of PKC subspecies with closely related structures. The PKC isozymes α, β, and γ consist of four conserved (C1–C4) and five variable regions (V1–V5). The C1 region contains cysteine-rich zinc finger-like motifs that are immediately preceded by an autoinhibitory pseudosubstrate sequence and contains the recognition site for phosphatidylserine, DAG, and phorbol ester. The C2 region of some PKC isoforms is rich in acidic residues and contains the binding site for Ca2+. The C3 and C4 regions constitute the ATP- and substrate-binding lobes of the PKC molecule [124126] (Figure 6.1).

Figure 6.1. Structure of PKC isoforms.

Figure 6.1

Structure of PKC isoforms. PKC is composed of four conserved (C1–C4) and five variable (V1–V5) regions. C1 region contains binding sites for DAG, phorbol ester, phosphatidylserine, and the PKC antagonist calphostin C. C2 region contains (more...)

PKC isoforms are classified into three groups. The conventional PKCs α, βI, βII, and γ have the four conserved regions (C1–C4) and the five variable regions (V1–V5). The novel PKCs δ, ϵ, η(L), and θ lack the C2 region and therefore do not require Ca2+ for activation. The atypical PKCs ζ and λ/ι have only one cysteine-rich zinc finger-like motif and are dependent on phosphatidylserine, but not affected by DAG, phorbol esters, or Ca2+ (Figure 6.1).


When PKC is not catalytically active, the basic autoinhibitory pseudosubstrate is protected from proteolysis by an acidic patch in the substrate-binding site (Figure 6.2). When PKC is activated, it phosphorylates arginine-rich protein substrates, which neutralize the acidic patch and displace the pseudosubstrate from its binding site in the kinase core [126,127]. The amino acid sequence near the substrate phosphorylation site may assist in PKC substrate recognition. PKC isotypes show specificity in substrate phosphorylation. While α-, β-, and γ-PKC are potent histone kinases, δ-, ϵ-, and η-PKC have a poor capacity to phosphorylate histone IIIS [125].

Figure 6.2. Mechanisms of PKC activation.

Figure 6.2

Mechanisms of PKC activation. The inactive PKC molecule is folded in such a way to have an endogenous pseudosubstare bind to the protein kinase region. PKC activation by phosphatidylserine (PS), DAG or phorbol ester, and Ca2+ allows the PKC molecule to (more...)

PKC causes phosphorylation of membrane-bound regulatory proteins in VSM. MARCKS (myristoylated, alanine-rich C kinase substrate), a major PKC substrate, is bound to F-actin and may function as a cross-bridge between cytoskeletal actin and the plasma membrane [128]. Also, PKC causes phosphorylation of the inhibitory GTP-binding protein Gi, facilitating the dissociation of the αi subunit from adenylyl cyclase and thereby relieves it from inhibition [125].

PKC also affects plasma membrane channels and pumps. PKC inhibits BKCa channel activity in pulmonary VSM [129]. Also, thromboxane A2-induced inhibition of voltage-gated K+ channels and pulmonary vasoconstriction may involve ζ-PKC [130]. PKC may also phosphorylate and activate plasmalemmal or saroplasmic reticulum Ca2+-ATPase, an action that promotes Ca2+ extrusion and may explain the transient nature of the agonist-induced increase in VSM [Ca2+]i. In addition, the α1 subunit of Na+/K+-ATPase may serve as a PKC substrate. Furthermore, activated PKC may phosphorylate and activate the Na+/H+ antiport exchanger and thereby increase the cytoplasmic pH [131].

PKC also phosphorylates regulatory proteins in VSM cytoskeleton and contractile myofilaments. PKC phosphorylates vinculin, a cytoskeletal protein localized at adhesion plaques, thus controlling cell shape and adhesion. PKC also phosphorylates CPI-17, which in turn inhibits MLC phosphatase, increases MLC phosphorylation, and thereby enhances VSM contraction [132]. The 20-kDa MLC and MLCK serve as substrates for PKC, and their phosphorylation could counteract the Ca2+-induced actin–myosin interaction and force development [133]. On the other hand, activation of α-PKC could cause phosphorylation of calponin, an actin-associated regulatory protein, and thereby enhance VSM contraction [125]. A specific link likely exists between each PKC isoform and one or more specific substrates in VSM, and identification of these specific interactions needs to be further examined.


PKC isoforms are expressed in different proportions in VSM of various vascular beds (Table 6.1). α-PKC is a universal isoform that is expressed in almost all blood vessels tested. γ-PKC is mainly expressed in the neurons and vascular nerve endings. δ-PKC is mainly associated with the vascular cytoskeleton. ζ-PKC is a universal isoform that has been found in many tissues. η/L-PKC has been found in the lung, skin, heart, and brain. θ-PKC is mainly expressed in skeletal muscle, while ι/λ-PKC is expressed in the ovary and testis [125].

Table 6.1

Table 6.1

Subcellular Distribution of PKC


The PKC isoforms α, β, and γ are mainly localized in the cytosolic fraction of unstimulated cells and undergo translation to the cell membranes in activated cells (Table 6.1). δ-PKC is located almost exclusively in the particulate fraction of both resting and activated cells. While ζ-PKC is localized near the nucleus of resting and activated mature VSMCs [143], it could also play a role in pulmonary vasoconstriction in the perinatal period [144].


An important question is what causes PKC to translocate. Simple diffusion may provide the driving force, while targeting mechanisms could allow high-affinity binding when PKC is near its target [143]. Targeting mechanisms may involve one of the following:

6.5.1. Conformation-Induced Changes in Hydrophobicity

Binding of Ca2+ or DAG may cause conformational changes in the PKC molecule that could result in exposure of the pseudosubstrate region, increase the hydrophobicity of PKC, and facilitate its binding to membrane lipids [126].

6.5.2. Lipid Modification

Lipid modification of proteins changes their subcellular distribution. Myristoylation of MARCKS is required for its binding to actin at the plasma membrane. PKC-mediated phosphorylation of MARCKS causes its displacement from the membrane and interferes with its actin cross-linking. Dephosphorylation of MARCKS causes its reassociation with the membrane through its stably attached myristic acid membrane-targeting moiety [145].

The architecture of VSM plasma membrane appears to be regulated. VSM sarcolemma is divided into domains of focal adhesions alternating with caveolae-rich zones, both harboring a subset of membrane-associated proteins. Likewise, sarcolemmal lipids are segregated into domains of cholesterol-rich lipid rafts and glycerophospholipid-rich nonraft regions. The segregation of membrane lipids is critical for preservation of membrane protein architecture and for translocation of proteins to the sarcolemma. In smooth muscle, membrane lipid segregation is supported by annexins that target membrane sites of distinct lipid composition, and each annexin requires different [Ca2+] for its translocation to the sarcolemma, and thus allows a spatially confined, graded response to external stimuli and intracellular PKC [146].

6.5.3. Phosphorylation

The change in electric charge caused by phosphorylation of the protein may affect its affinity for lipid. For example, phosphorylation of MARCKS has an electrostatic effect of equal importance to myristoylation in determining the protein affinity to the membrane. Also, phosphorylation of PKC itself may be required for its activation and translocation. The PKC phosphorylation sites appear to be located in the catalytic domain of α-, β-, and δ-PKC [147].

6.5.4. Targeting Sequences

Binding sites for arginine-rich polypeptides have been identified in the PKC molecule distal to the catalytic site and may allow targeting of PKC to specific subcellular locations. Also, receptors for activated C-kinase (RACKs) may allow targeting of PKC to cytoskeletal elements, and a peptide inhibitor derived from the PKC-binding proteins annexin I and RACKI may interfere with translocation of p-PKC [148].


PKC plays an important role in the cell adjustment to the environment by exerting both positive and negative effects on cellular events. PKC has been associated with numerous physiological functions, including secretion and exocytosis, modulation of ion conductance, gene expression, and cell proliferation [124,125]. PKC may also exert negative-feedback control over cell signaling via down-regulation of surface receptors and inhibition of agonist-mediated phosphoinositide hydrolysis [124]. Several studies suggest a role for PKC in VSM contraction [124,125,149151]. PKC activation by phorbol esters has been shown to cause significant contraction in isolated vascular preparations [125,149]. Also, PKC inhibitors cause significant inhibition of agonist-induced vascular contraction [150,151]. However, some studies suggest that PKC-mediated phosphorylation of MLCK may cause vascular relaxation [133].


PKC isoforms respond differently to Ca2+, phosphatidylserine, DAG, and other phospholipid degradation products. PKC binds Ca2+ in a phospholipid-dependent manner, and Ca2+ may form a “bridge” holding the protein and phospholipid complex together at the membrane [152]. Phosphatidylserine is indispensable for activation of PKC. Phosphatidylinositol and phosphatidic acid activate PKC at high Ca2+ concentrations. DAG activates PKC by reducing its Ca2+ requirement and enhancing its membrane association [124].

PKC activators also include lipids derived from sources other than glycerolipid hydrolysis such as cis-unsaturated free fatty acids and lysophosphatidylcholine, ceramide (a sphingomyelinase product), phosphatidylinositol 3,4,5-trisphosphate, and cholesterol sulfate [153]. Phorbol esters such as TPA, PMA, and PDBu can substitute for DAG in PKC activation. Phorbol esters stabilize PKC–membrane association by reducing its apparent Km for Ca2+ [125].

Autophosphorylation of PKC may modify its activity or affinity for its substrates. α-, βI-, and βII-PKC are synthesized as inactive precursors that require phosphorylation by a putative “PKC kinase” for permissive activation. Also, multiple phosphorylation of α-PKC prevents its down- regulation by phorbol esters. Phosphorylation at the extreme C-terminus of βII-PKC allows the active site to bind ATP and substrate with higher affinity, while phosphorylation of structure determinants in the regulatory region enable higher affinity binding of Ca2+ [154].


The role of PKC in VSM contraction has been verified by the use of PKC inhibitors. Several PKC inhibitors have been developed. PKC inhibitors acting in the catalytic domain compete with ATP and therefore may not be specific. PKC inhibitors acting in the regulatory domain compete at the DAG/phorbol ester or the phosphatidylserine binding site and may be more specific. Extended exposure to phorbol esters can specifically down-regulate α-, β-, and γ-PKC, but the tumor-promoting properties of phorbol esters limit their use.

The regulatory domain of PKC contains an amino acid sequence between residues 19 and 36 that resembles the substrate phosphorylation site. Synthetic oligopeptides based on pseudosubstrate sequence are specific PKC inhibitors because they exploit its substrate specificity and do not interfere with ATP binding. The synthetic peptide (19–36) inhibits both PKC autophosphorylation and protein substrate phosphorylation. Replacement of Arg-27 with alanine in the peptide [Ala-27]PKC (19–31) increases the IC50 for inhibition of substrate phosphorylation [127]. Also, a myristoylated peptide based on the substrate motif of α- and β-PKC, myr-ΨPKC, inhibits TPA-Induced PKC activation and phosphorylation of MARCKS [155]. In smooth muscle, α-tocopherol inhibits the expression, activity, and phosphorylation of α-PKC. Interestingly, β-tocopherol protects PKC from the inhibitory effects of α-tocopherol [156].

siRNA for specific PKC isoforms is now available and should be useful for studying the role of PKC in various cell functions. Also, antisense techniques, transgenic animals, and knockout mice have been useful in studying the effects of PKC down-regulation in vivo.


The interaction of a PKC isoform with its protein substrate may trigger a cascade of protein kinases that ultimately stimulate VSM contraction. PKC may phosphorylate CPI-17, which in turn inhibits MLC phosphatase, increases MLC phosphorylation, and enhances VSM contraction (see Figure 1.1) [132]. PKC may also phosphorylate the actin-binding protein calponin, and thereby reverses its inhibition of actin-activated myosin ATPase, allows more actin to interact with myosin, and increases VSM contraction (Figure 1.1) [2].

PKC, MAPK, and c-Raf-1 have been implicated in VSM growth. MAPK is a Ser/Thr kinase that is activated by dual phosphorylation at Thr and Tyr residues. In quiescent undifferentiated cultured VSMCs, MAPK is mainly cytosolic but translocates to the nucleus during activation by mitogens. Tyrosine kinase and MAPK activities have also been identified in differentiated contractile VSM [143,157]. MAPK transiently translocates to the surface membrane during early activation of VSM but undergoes redistribution to the cytoskeleton during maintained VSM activation [157] (Figure 6.3). It has been suggested that during VSM activation, DAG promotes translocation of cytosolic ϵ-PKC to the surface membrane, where it is fully activated. Activated ϵ-PKC stimulates the translocation of cytosolic MEK and MAPK to the plasmalemma, where they form a surface membrane kinase complex. PKC causes phosphorylation and activation of MEK, which in turn phosphorylates MAPK at both Thr and Tyr residues. Tyrosine phosphorylation targets MAPK to the cytoskeleton, where it phosphorylates the actin-binding protein caldesmon and reverses its inhibition of MgATPase activity and thus increases actin–myosin interaction and VSM contraction (Figure 6.3) [2,157].

Figure 6.3. Protein kinase cascade leading to VSM contraction.

Figure 6.3

Protein kinase cascade leading to VSM contraction. Agonist (A)-induced activation of VSM receptor (R) leads to generation of DAG and translocation of PKC to the surface membrane (A). Other kinases such as Raf, MEK, and MAPK follow PKC and form a membrane (more...)

Copyright © 2010 by Morgan & Claypool Life Sciences.
Bookshelf ID: NBK54587


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